Engineering of a P450-based Kemp Eliminase with a New Mechanism

For three decades the biocatalytic version of the Kemp elimination of 5-nitro-benzisoxazole (1) has served as a forum for testing the creation of different artificial enzymes, the primary aim being to reveal mechanistic intricacies and to extend our understanding of enzymatic catalysis as such. In general, acid/base catalysis pertains, but recently a novel redox based mechanism was postulated when using P450-BM3 mutants as scaffolds. In the present study, we report an surprising discovery made upon employing new P450-BM3 variants generated by rational enzyme design, which points to the existence of a new and different redox based mechanism. X-ray structural data and theoretical analyses based on MD simulations and QM/MM calculations support this conclusion. The results of this study are of relevance in the human metabolism of therapeutic drugs and in redox mediated biosynthesis catalyzed by P450s.